A wide range of biological responses are mediated by two families of GTP-binding (G) proteins, the trimeric G proteins that couple seven-membrane spanning (heptahelical) receptors to the generation of key cellular second messengers, and the Ras superfamily of single chain GTP-binding proteins (designated small G proteins) that are activated by guanine nucleotide exchange factors (GEFs). The investigators have used the rhodopsin/transducin-couple phototransduction system as a model for developing fluorescence spectroscopic approaches to monitor directly each step in a G protein-mediated signaling pathway. Recently, they have developed similar fluorescence readouts for the small G protein Cdc42. In this renewal application, they intend to take advantage of these spectroscopic assays, together with the availability in the laboratory of recombinant expression systems for the transducin-a subunit (aT), Cdc42, and various target and regulatory proteins, to determine the molecular mechanisms by which G proteins are activated and how these activation events are converted into the regulation of target/effector activities. Three main lines of research will constitute the specific aims of this proposal. 1. Molecular mechanisms of G protein activation. Here the investigators will determine and compare the fundamental mechanisms underlying the activation of Cdc42 by its GEF, the Dbl oncoprotein, and the activation of aT by rhodopsin. 2. Identification and characterization of novel dominant-active and dominant-negative G proteins. Here the investigators will extend earlier findings to determine whether mutations within the conserved guanine ring-binding motif of trimeric G protein a subunits yield novel dominant-negative mutants as well as examine why the mutation of a glutamic acid residue at position 203 in the aT subunit yields a constitutively active G protein, despite the fact that it is fully active as a GTPase. These studies should provide important insights into the switch mechanisms that underlie G protein function as well as provide valuable reagents for studying new types of G protein-signaling pathways. 3. Mechanisms of G protein-mediated target regulation. They will identify the different regions on a trimeric G protein a subunit (aT) that are involved in binding a target/effector molecule (gPDE) and a new class of regulators (RGS molecules) and determine how these binding interactions are converted into the regulation of target/effector activity. Overall, the proposed studies should be relevant to a variety of biological response pathways including the regulation of adenylyl cyclase activity, vertebrate vision, and cell growth and differentiation.